Abstract

Transmission electron microscopy (TEM) analysis of ciliary ultrastructure
is classically used for the diagnosis of primary ciliary dyskinesia (PCD).
We report our extensive experience of TEM analysis in a large series of patients
in order to evaluate its feasibility and results.

TEM analysis performed in 1,149 patients with suspected PCD was retrospectively
reviewed. Biopsies (1,450) were obtained from nasal (44%)
or bronchial (56%) mucosa in children (66.5%)
and adults (33.5%).

TEM analysis was feasible in 71.4% of patients and showed a main
defect suggestive of PCD in 29.9%. TEM was more feasible in adults
than in children, regardless of the biopsy site. Main defects suggestive of
PCD were found in 76.9% of patients with sinopulmonary symptoms and
in only 0.4% of patients with isolated upper and 0.4% with isolated
lower respiratory tract infections. The defect pattern was similar in children
and adults, involving dynein arms (81.2%) or central complex (CC) (18.8%).
Situs inversus was never observed in PCD patients with CC defect. Kartagener
syndrome with normal ciliary ultrastructure was not an exceptional condition (10.2%
of PCD).

In conclusion, TEM analysis is feasible in most patients and is particularly
useful for PCD diagnosis in cases of sinopulmonary syndrome of unknown origin.

Cilia, evolutionarily conserved structures, are classified according to
their cytoskeleton core called axoneme: primary cilia with sensory function,
and motile cilia ensuring fluid transport. Defects in primary cilia have been
associated with a growing number of rare genetic diseases (polycystic
kidney disease, Bardet–Biedl syndrome and retinitis pigmentosa),
whereas motile cilia are involved in the most prominent ciliopathy called
primary ciliary dyskinesia (PCD) 1.

PCD is a congenital disorder with an estimated prevalence of 1:15–30,000
live births 2 and is due to impaired
mucociliary transport resulting from a lack of ciliary motion leading to chronic
respiratory infections. The clinical features of PCD, usually beginning in
early childhood, are characterised by bronchiectasis and chronic sinusitis,
sometimes associated with situs inversus and male sterility 3. The axoneme of motile cilia is composed of nine
peripheral doublet microtubules with attached inner and outer dynein arms (IDA
and ODA, respectively) and radial spokes, surrounding a central complex (CC)
consisting of two central microtubules surrounded by the central sheath. PCD
is a heterogeneous group of genetic disorders usually transmitted as autosomal
recessive traits with various ciliary ultrastructural defects 3. The absence of pathognomonic clinical and laboratory
signs makes PCD difficult to diagnose. However, it is of prime importance
to recognise this disease in order to start appropriate therapy of respiratory
tract infections and minimise lung damage. In this context, the finding by
Afzelius 4 that most respiratory
cilia of patients with PCD carry ultrastructural defects has opened up new
ways to manage this disease, especially by providing the first objective test
of diagnostic value.

Transmission electron microscopy (TEM) analysis of cilia is still
a relevant technology now frequently combined with new innovative investigations
for the diagnosis of PCD. However, TEM analysis is an arduous and expensive
technique performed in highly selected patients, requiring airway biopsies
that are processed by multiple technical steps. More importantly, TEM can
be defective for analysis of ciliary ultrastructure even after optimal processing.
Few studies based on large series of patients (i.e. >50 patients)
with PCD have been reported 5–7 and none of them have focused on the efficacy
of TEM analysis of cilia. It therefore seemed important to report our lengthy
experience of TEM analysis of cilia based on a large series of patients. The
feasibility and results of TEM analysis of cilia performed in our institution
from 1985 to 2006 were retrospectively analysed in the present study.

PATIENTS AND METHODS

Patients

Physicians experienced in respiratory endoscopy from several hospitals
consecutively sent airway biopsies to our laboratory and these samples were
consecutively examined for TEM analysis of cilia. Airway biopsies were performed
in patients with respiratory tract infections suggestive of PCD in two main
clinical situations: 1) highly suggestive presentation of PCD (i.e. situs inversus with airway infections); and 2) recurrent
airway infections (i.e. rhinosinusitis, bronchitis, bronchiectasis)
after exclusion of all known pathological conditions (such as cystic
fibrosis or immunodeficiency). The physicians performed biopsies after
at least 30 days absence of exacerbation of respiratory tract infection
and, if necessary, at the end of an antibiotic course.

Ciliary ultrastructure

Biopsies were obtained from either bronchial (main bronchus)
or nasal (inferior turbinate) mucosa of children (<18 yrs
of age) or adults. Children with suspected PCD were essentially referred
to paediatric respiratory physicians who mainly performed bronchial biopsies,
whereas adults with suspected PCD were essentially referred to ENT physicians
who performed nasal biopsies. Patients or their parents were informed by their
physicians of the exact nature and goal of all investigations performed and
gave their informed consent.

Airway biopsies were immersed in 2.5% glutaraldehyde and processed
as usual for ultrastructural analysis 8.
Ultrathin sections were examined at a final magnification of ×60,000
without knowledge of the clinical data. In each specimen, analysis of at least
50 transverse ciliary sections of different cells was required to study the
internal axonemal structure according to a quantitative method 9. Ciliary ultrastructure results were expressed
as a percentage of abnormal cilia among the total number of cilia analysed.
As previously reported, up to 10% of cilia in control specimens can
exhibit ultrastructural defects 10, 11. For this study, ciliary abnormalities
were defined as the presence of >20% of ciliary defects. For each
ciliary study, axonemal abnormalities were quantified and expressed as a percentage
of each ultrastructural defect over the total number of abnormal cilia to
define the main ultrastructural defect. The main ultrastructural defect can
concern ODA (total absence or short ODA isolated or associated with absence
of IDA), IDA (absence of IDA isolated or associated with radial
spoke defect) or CC (central microtubules absent or single)
abnormalities. Since 2002, for questionable IDA in micrographs obtained by
TEM, computerised analysis of cilia was systematically performed to improve
IDA visualisation, as previously reported 12. Ciliary orientation was systematically evaluated by comparing
the position of the central pairs of adjoining cilia as previously described 11, 13. Disorientation was defined as an angle >25°.

Statistical analysis

Results are expressed as numbers and percentages for categorical data,
and as median with interquartile range (IQR) and range for quantitative
data with a non-normal distribution. Main results were expressed with their
95% confidence interval (CI). Comparisons of categorical
data were performed with Chi-squared test or Mantel–Haenszel statistics
when adjusted on a third variable and quantitative data were compared by Kruskal–Wallis
nonparametric test. The relationship between site of infection and main defect
was tested by Chi-square test. A p-value <0.05 was considered significant.

RESULTS

TEM feasibility

1,149 patients (corresponding to 1,450 biopsies) were examined
for ciliary ultrastructure. TEM was unfeasible (see below) in 329
patients (28.6%) and feasible in 820 patients (71.4%),
constituting the study population. Among the 329 patients in whom TEM was
unfeasible (corresponding to 464 (32%) out of 1,450
biopsies), 207, 111 and 11 patients were biopsied once, twice or three
or more times, respectively. The first biopsy did not provide samples of sufficient
quality for TEM analysis in 459 patients (39.9%). Among these
patients, 252 (54.9%) received a second biopsy providing
samples of sufficient quality in 98 patients (38.9%). Finally,
among the 154 patients with unfeasible TEM in the second sample, 43 (27.9%)
received a third biopsy providing samples of sufficient quality for TEM in
32 (74.4%) patients.

We found that feasibility of TEM analysis did not vary with year (table 1⇓). Among the 1,450 airway biopsies,
636 (44%) were obtained from nasal mucosa and 814 (56%)
from bronchial mucosa. TEM was significantly more feasible on nasal (458/636,
72%) than on bronchial (528/814, 64.8%) biopsies (p<0.03).
Among these 1,450 biopsies, 965 (66.5%) were performed in
children and 485 (33.5%) were performed in adults. TEM was
significantly more feasible in biopsies from adults (424/485, 87.4%)
than in biopsies from children (562/965, 58.2%) (p<0.001).
Within the children's group, the TEM was significantly more feasible
in the 10–17-yr-old range than in the 0–9-yr-old range (p<0.05) (table 1⇓). Nasal biopsies were performed more
frequently in adults (322/485, 66.4%) and bronchial biopsies
were performed more frequently in children (651/965, 67.5%).
The difference of TEM feasibility between nasal and bronchial biopsies was
no longer observed when adjusted for the patient's age (p = 0.33),
while the difference between adults and children persisted when adjusted for
biopsy site (p<0.001).

TEM analysis showed normal ciliary ultrastructure (group I) in
533 patients (65%) (fig. 1a⇓), heterogeneous ciliary abnormalities without
a main ultrastructural defect (group II) in 15 patients (1.8%),
abnormal cilia with a main ultrastructural defect (group III) in
245 patients (29.9%) and questionable ciliary ultrastructure (group
IV) in 27 patients (3.3%) even after computerised analysis
of cilia. In fact, computer-assisted analysis of micrographs, performed for
95 patients, allowed us to reach a conclusion for 68 patients (15 and
53 patients belonging to groups I and III, respectively). The median,
IQR and range of percentage of abnormal cilia in each group are given in table 2⇓.

Characteristics of the groups as defined by ciliary ultrastructure
in the 820 patients with feasible transmission electron microscopy analysis

The children/adults ratio was significantly different between groups (p<0.001)
corresponding to a higher proportion of children in group III (table 2⇑). Although present in each group,
situs inversus was significantly more frequent in group III (31.8%)
than in the other groups (p<0.0001) (table 2⇑).

Although only limited clinical information was provided by most of the
physicians who performed the biopsies, the topography of upper and/or
lower airway infections could be studied in the various groups. Interestingly,
90% of patients in group I and II suffered from chronic infections
exclusively involving either upper or lower airways (table 2⇑). In contrast, 99.2% of patients
in group III suffered from a combination of upper and lower airway infections, i.e. sinopulmonary syndrome that was significantly more frequent in this
group than in the other groups (p<0.001) (table 2⇑). A main defect suggestive of PCD
was found in 243 (76.9%) of the 316 patients with sinopulmonary
syndrome and in two (0.4%) of the 504 patients without sinopulmonary
syndrome. These two patients were a child with severe asthma without upper
airway infection, and an adult with situs inversus and nasal polyposis without
lower airway infection. In group I, 28 patients with normal ciliary ultrastructure
had the clinical criteria to diagnose Kartagener syndrome (i.e.
association of sinusitis, bronchiectasis and situs inversus) and we considered
them as Kartagener syndrome with normal ciliary ultrastructure.

In group I, the percentage of abnormal cilia was very low, as previously
reported in controls 10. In
group II, the heterogeneous ciliary abnormalities mostly concerned the peripheral
microtubules. In group III (table 3⇓),
the main ultrastructural defect concerned ODA in 64.9% of patients,
IDA alone in 16.3% of patients and CC in 18.8% of patients.
Situs inversus was significantly more frequent in patients with ODA than in
patients with IDA defects and was never observed in patients with CC abnormalities (p<0.001).
The median, IQR and range of abnormal cilia in patients with ODA, IDA and
CC defects are given in table 3⇓.
ODA abnormalities (n = 159) were either isolated (total
absence or short ODA) in 81 patients (fig. 1b⇑) or associated with absence of IDA in 56 patients (fig. 1c⇑); in the remaining 22 patients, the
ODA defect was associated with questionable IDA (fig. 1d⇑). Absence of IDA alone (n = 40)
was either isolated (nine patients) (fig. 1e⇑) or associated with radial spoke defect (31
patients) (fig. 1f⇑).
Interestingly, in group III, only 142/245 patients (58%)
exhibited 100% of abnormal cilia and the abnormalities always involved
the dynein arms and never the CC (table 3⇓). The main ultrastructural defect in children
from group III concerned ODA, IDA and CC for 61.2%, 18.2% and
20.6% patients, respectively. The main ultrastructural defect in adults
from group III concerned ODA, IDA and CC for 72.5%, 12.5% and
15% patients, respectively.

Phenotypic features according to the main ciliary defects as defined
by transmission electron microscopy(group III, n = 245)

In group IV, with questionable ultrastructure, even after computerised
analysis of cilia, most difficulties concerned analysis of IDA (24/27
patients, 88.9%) (fig. 1g⇑),
whereas analysis of ODA was questionable in only three out of 27 patients (11.1%)
and CC analysis was never a problem (fig. 1h⇑).

The results of ciliary orientation remained within the normal range in
the four groups and were not related to ultrastructural abnormalities (data
not shown).

DISCUSSION

We report our 20-yr experience of TEM analysis based on a historical series
of >1,000 patients with suspected PCD, indicating the limitations of the
technique that was not feasible in nearly one-third of patients, but also
demonstrating that TEM remains a useful tool for the diagnosis of PCD, providing
a precise ultrastructural phenotype in most cases. Our study highlights that
PCD with CC defects and Kartagener syndrome with normal ciliary ultrastructure
are not exceptional conditions. TEM analysis is particularly useful in the
case of sinopulmonary syndrome of unknown origin, especially for identification
of PCD with CC defects, a condition that is never associated with situs inversus.

In our series of 1,450 airway biopsies, the TEM failure rate was comparable
with that of previous studies 14, 15. Epithelial metaplasia and denuded basement
membrane were the main reasons for failure in nasal and bronchial biopsies,
respectively. The site of biopsy for ciliary studies has rarely been studied
in the literature, except for one paediatric study that also showed a high
frequency of metaplasia in nasal biopsies 16. Analysis showed a similar TEM feasibility between nasal and
bronchial biopsies, regardless of the age. Interestingly, TEM analysis was
significantly more feasible in biopsies from adults than in biopsies from
children, probably due to the technical challenge of taking biopsies of the
narrow airways of children, accounting for the small size of biopsies. In
fact, when TEM analysis was unfeasible after the first sampling, the repetition
of biopsies seemed to be helpful and the interval between biopsies appeared
to be less important than the absence of exacerbation of respiratory tract
infections.

In the 820 patients in whom TEM analysis was feasible, ciliary ultrastructure
was normal in more than one-half of patients, which raises the issue of accurate
patient selection for TEM analysis of cilia. Situs inversus associated with
airway infections is the most suggestive condition justifying systematic TEM
analysis of cilia. However, situs inversus is not constant in PCD 5–7, 17 and ciliary
studies are often proposed in the presence of upper and/or lower chronic
airway infections of unknown origin. The strong relationship between the presence
of a sinopulmonary syndrome and the detection of a main defect suggests that
this syndrome could be a good clinical criterion to improve patient selection
for TEM analysis of cilia. However, all other known pathological conditions
must be excluded before performing TEM analysis, which may be unfeasible in
about one-third of these patients.

In line with the literature, the main ciliary defect (group III)
concerned dynein arms in >80% of cases and more frequently concerned
ODA than IDA in adults and in children 6, 7. Our results,
based on the study of a very large series of patients, describe a spectrum
of ultrastructural defects in PCD similar to those reported in other studies (i.e. isolated ODA defects: 24–43%; ODA associated with IDA
defects: 24–45%; isolated IDA defects: 14–29%; CC
defects: 4–18%) 6, 7, 18. In most patients with a main ciliary defect, all cilia were
abnormal, as expected for a congenital disease. However, some normal cilia
can persist, rarely associated with dynein arm defects but always in the case
of CC defects 9, 19. Although ultrastructural defects are usually not
detected in more than half of the cilia, CC defects are considered to be congenital 3, 9, 20. The constitutional nature of this specific
defect is demonstrated by the existence of sibling forms of PCD with CC defects 20 and similar abnormalities described
in Chlamydomonas mutants, a cellular model for PCD 21–24.
In patients with CC defects, the presence of about one-half of normal cilia
can be explained by various hypotheses: 1) instability of central microtubules 25, 2) short length of central microtubules
only present in the basal part of the cilia 22, or 3) quantitative synthesis deficiency providing central
microtubule structures for only some cilia.

Patients with heterogeneous abnormalities of cilia (group II)
were uncommon; these abnormalities concerned the number of peripheral microtubules,
and are considered to be acquired ciliary defects related to recurrent airway
damage 20, 26. TEM analysis of cilia in cultured respiratory
epithelial cells has been proposed to eliminate such heterogeneous abnormalities
of cilia 18.

In very few patients (group IV), TEM analysis was feasible but
ultrastructure was questionable. As previously reported, IDA is the most frequent
questionable axonemal structure 19, 27 because of its low contrast on TEM.
The use of computer-assisted analysis of micrographs greatly improves dynein
arm visualisation in doubtful cases after classical TEM 12.

It is noteworthy that situs inversus was observed in <50% of
patients with a main ciliary defect. This result, in agreement with previous
data 6, is lower than the classically
reported proportion of situs inversus 5, 7, 17. This discrepancy could be due to patient selection mainly based
on the presence of chronic airway infections of unknown origin, but not necessarily
associated with situs inversus. Finally, our study showed that the proportion
of situs inversus varies with the type of ultrastructural defect, is more
frequent in the case of abnormal ODA than IDA, and is never associated with
CC defects, as already mentioned 6, 20. The absence of situs inversus in the
case of CC defects could be explained by the fact that visceral lateralisation
is initiated by nodal cilia that normally do not contain a central pair of
microtubules. It is therefore not surprising that a molecular defect involving
the central microtubules does not modify visceral lateralisation.

Finally, Kartagener syndrome with normal cilia was not an exceptional condition,
found in 28 of 109 patients with situs inversus. Similarly, based on random
lateralisation, it can be speculated that our series should contain the same
proportion of “true” patients with PCD but with normal cilia and
without situs inversus. Strikingly, 25 patients from group I with
normal cilia (and without situs inversus) exhibited sinopulmonary
syndrome. Overall, an estimated 18% ((28+25 = 53)/(28+25+245 = 298))
of patients with PCD may exhibit normal cilia. Due to the absence of ciliary
ultrastructural defects in these patients, the diagnosis of PCD requires additional
diagnostic tools.

Due to the long observation period, new diagnostic tools have become available
during that time that enhance patient selection for TEM analysis and/or
improve diagnostic procedures. Considering the limitations of TEM analysis
stressed in this study (i.e. unfeasible in nearly 30%
of the patients and Kartagener syndrome with normal ciliary ultrastructure),
the diagnosis of PCD now relies on the association of TEM analysis and these
tools 2, 28. In addition to the evaluation of ciliary beat
frequency, which frequently remains the first investigation for the diagnosis
of PCD 29, high-resolution,
digital high-speed videomicroscopy has been developed to characterise abnormal
beat patterns specific for axonemal defects 6, 7, 30. Nasal nitric oxide is dramatically
reduced in most patients with confirmed PCD 7, 31. Immunostaining
methods using antibodies directed against the main axonemal components are
also developed to facilitate identification of structural abnormalities of
cilia 32. Lastly, identification
of genes involved in the pathogenesis of PCD would be an ideal tool. This
approach remains challenging because PCD is genetically heterogeneous with
numerous candidate genes that are sometimes very large. Two main genes, DNAI1 and DNAH5 have been identified to date 33, 34
for PCD with isolated ODA defects and the other identified genes (i.e.RPGR, TXNDC3, DNAH1, DNAI2, KTU, RSPH9 and RSPH4A) concern a few PCD families 35–40. The identification of the ciliary ultrastructural defect by
TEM analysis could also be helpful for genetic analysis.

In conclusion, TEM is often feasible and very useful in defining the ultrastructural
phenotype, particularly in the case of sinopulmonary syndrome of unknown origin.
In patients with isolated upper or lower respiratory tract infections, TEM
should be performed only if there is a very high level of suspicion. TEM is
especially helpful for the diagnosis of PCD when situs inversus is absent
as we constantly found in PCD with CC defects. Our results also highlight
that the spectrum of ultrastructural defects is similar in children and adults
and that Kartagener syndrome with normal ciliary ultrastructure is not an
exceptional condition, representing a technical challenge for the diagnosis
of PCD. Our study also stresses the limitations of solely using ultrastructural
analysis in PCD diagnosis, which is currently based on convergent elements
derived from clinical phenotype, TEM results and additional diagnostic tools,
and is essential to start early appropriate therapy designed to prevent lung
damage.

Support statement

This work was supported by grants from the Legs Poix from the Chancellerie
des Universites, the Assistance Publique-Hopitaux de Paris (PHRC AOM06053,
P060245) and the Agence Nationale pour la Recherche (ANR-05-MRAR-022-01).

Statement of interest

None declared.

Acknowledgments

The authors are grateful to the patients and their families who participated
in this study. We would also like to thank B. Escudier for careful review
and useful advice for writing this manuscript.

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